Shiraz E Med J. 15(12): e7379.
DOI: 10.5812/ircmj.7379
Research Article
Published online 2013 December 5.
Ultrastructure of in vitro Matured Human Oocytes
1
2,*
2
Abbas Shahedi , Mohammad Ali Khalili , Mehrdad Soleimani , Shekoufeh Morshedizad
3
1 Department of Anatomy, Yazd Branch, Islamic Azad University, Yazd, IR Iran
2Yazd Institute for Reproductive Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, IR Iran
3Islamic Azad University, Yazd, IR Iran
* Corresponding authors: Mohammad Ali Khalili, Yazd Institute for Reproductive Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, IR Iran, Tel: +98-3518247088,
Fax: +98-3518247087, E-mail: [email protected]; Abbas Shahedi, Department of Anatomy, Yazd Branch, Islamic Azad University, Yazd, IR Iran. Tel: +98-3518210542, Fax: +983518210542, E-mail: [email protected]
Received: July 23, 2012; Revised: June 23, 2013; Accepted: October 26, 2013
Background: Approximately 20% of recovered oocytes are immature and discarded in intracytoplasmic sperm injection (ICSI) procedures.
These oocytes represent a potential resource for both clinical and basic science application.
Objective: The aim of this study was to evaluate the ultrastructure architecture of in vitro matured human oocytes using transmission
electron microscopy (TEM).
Materials and Methods: A total of 204 immature oocytes from infertile patients who underwent ICSI cycles were included in this
prospective study. Immature oocytes were divided into two groups: (i) GV oocytes (n = 101); and (ii) MI oocytes (n = 103). Supernumerary
fresh in vivo matured oocytes (n = 10) were used as control.
Results: The rates of maturations were 61.38% for GV and 73.78% for MI oocytes in IVM medium (P = 0.07). However, the rate of oocyte arrest
was significant between groups (P <0 .05). Ultrastructurally; in vitro and in vivo matured oocytes appeared round, with a homogeneous
cytoplasm, an intact oolemma and an intact zona pellucida. However, immature oocytes indicated numerous large mitochondria-vesicle
complexes (M-VC).
Conclusions: Ultrastructural changes of M-VC in IVM groups emphasize the need for further research in order to refine culture conditions
and improve the implantation rate of in-vitro matured oocytes.
Keywords: In vitro Maturation; Germinal Vesicle Oocytes; Metaphase-I Oocytes; Ultrastructure
1. Background
Approximately 20% of aspirated oocytes following hormone stimulation are immature at the germinal vesicle
(GV) or metaphase I (MI) stage. These oocytes are discarded due to their reduced potential for embryo development under current culture conditions (1). However, this
cohort of oocytes is benefit for studies aimed at elucidating the mechanisms of in vitro maturation of human
oocytes and might ultimately contribute to the pool of
embryos available for embryo transfer (2, 3). It would be
clinically useful if the in-vitro maturation rates of these
oocytes could be improved because some patients produce only a few oocytes, while others may produce mostly immature oocytes (4).
Although, healthy infants have been born following
IVM protocols. but, the pregnancy rates are significantly
lower when compared with in vivo matured oocytes in
gonadotrophin stimulated cycles (5). Thus, improving
the IVM success rate remains an important challenge to
obtain better efficiency for fertility preservation (6). IVM
has several advantages to reduce costs; avoiding the side
effects of ovarian hyperstimulation syndrome and simplifying treatment for certain infertile couples (7). The
ability to resume meiosis is independent of the cumulus cells (8). Thus, the oocyte is able to undergo nuclear
maturation without presence of cumulus and granulosa
cells. Clinical practice has shown a correlation between
morphological quality of IVM oocytes and fertilization,
cleavage and implantation rates. Routine evaluation of
oocyte morphology by phase-contrast microscopy (PCM)
is an important predictive marker of oocyte quality, currently utilized for evaluation of the success of a given ART
program (9). However, low-resolution morphological
assessment is not always a sufficient measure of oocyte
fertility potential and developmental competence (10).
Therefore, use of electron microscopy, integrated by other investigational approaches, seems especially effective
in evaluation of oocyte quality.
2. Objectives
The present study was therefore aimed to investigate
the ultrastructure of immature human oocytes obtained
Implication for health policy/practice/research/medical education:
In vitro maturation technique is relatively a safe procedure for human immature oocytes.
Copyright © 2013, Iranian Red Crescent Medical Journal; Published by Kowsar Corp. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Shahedi A. et al.
from ICSI cycles that were matured in vitro with transmission electron microscopy (TEM).
3. Materials and Methods
3.1. Study Population
This prospective study included 85 women aged 28-40
years who were consecutively treated with ICSI in Yazd institute for reproductive sciences. Informed consent was
obtained from each patient before the start of research.
Supernumerary MII oocytes from cases that were cancelled due to azoospermia on the day of oocyte retrieval
were used for TEM. Also, oocytes that were distinguished
to be at M-I or GV stages on day 0 were assigned for IVM.
Ovarian stimulation was induced with long protocols
using GnRH agonist down-regulation, followed by rFSH
(Gonal-F; Serono, Switzerland). The ovarian response was
controlled by transvaginal ultrasound and serum estradiol concentration on the day of hCG injection. When
diameter of at least two follicles was larger than 18 mm,
10,000 IU of hCG (i.m.; Profasi, Serono) was administered.
Oocyte pick-up was performed 36 h after hCG injection
under transvaginal ultrasound-guidance. All follicles
larger than 14 mm were subjected to pick-up, while follicles smaller than 14 mm were not punctured. There was
uniformity of the ultrasound measurements between our
physicians (11). After oocyte retrieval, the oocytes were denuded by a short exposure with 80 IU/ml hyaluronidase
(Sigma Co, St. Louis, Mo, USA) and mechanically by using
fine-bore glass pipettes. After 2-3 h, the oocytes were then
assessed for nuclear maturation (GV, MI or MII), under
the stereo microscope (Olympus Co, Tokyo, Japan) and
separated based on maturity. Those that were considered
mature underwent ICSI. This study was approved by the
ethical committee of the institute.
3.2. Groups
Group 1: The supernumerary mature oocytes were obtained from consenting ICSI patients (n = 10). Groups 2
and 3 were cases with immature oocytes at GV (n = 101)
and MI stage (n = 103), respectively. Immature oocytes in
groups 2 and 3 were transferred to pre-warmed IVM medium (3 oocytes per 50µl droplet under mineral oil) containing Hams F10 (Biochrom Co, Germany) supplemented with 0.75 IU LH, 0.75 IU FSH (Ferring Co, Germany) with
40% human follicular fluid (HFF). The oocytes were then
cultured in a 5% CO2 incubator at 37 °C with high humidity. After 36 h culture, the oocyte maturation was determined by the presence of the first polar body.
3.3. Electron Microscopy
A total of 30 mature oocytes were prepared for TEM.
From these oocytes, ten oocytes were matured in-vivo
and the rest were matured by IVM technology. Oocytes
were fixed and processed for TEM as described by Nottola
2
et al. (12). Oocytes were fixed in 1.5% glutaraldehyde (Sigma, Mo, USA) in 0.1 M phosphate buffer for 2-5 days at 4 °C,
and washed in the same buffer for 10 min, and post-fixed
in 1% osmium tetroxide (Agar Scientific, Stansted, UK) in
phosphate buffer, and washed again in the same buffer.
Oocytes were then embedded in small blocks of 1% agar
(Sigma, Mo, USA), dehydrated in increasing concentrations of ethanol, immerssion in propylene oxide for solvent substitution and individually embedded in Araldite
resin (Merk, Germany). The PH of both fixatives ranged
from 7.2 to 7.4 at room temperature. For light microscopy,
the oocytes were sectioned at a thickness ranging from
0.5 to 1 µm and stained with Toluidine blue, and then examined by light microscopy (Zeiss Axioskop). Ultrathin
sections (60 - 80 nm) were cut and stained with uranyl
acetate (7 min) and lead citrate (13 min). These sections
were observed and photographed with a TEM at 80KV
(Zeiss, Germany).
3.4. Statistical Analysis
Differences in oocyte maturational stages, between the
groups were calculated and compared using Chi-square
test. P < 0.05 was considered statistically significant. Calculations were performed by SPSS software (version 16,
USA).
4. Results
4.1. Oocyte Maturation
61.38% of GV and 73.78% of M-I stage oocytes were matured after 36h in IVM medium. There were significant
differences in rates of immature oocytes arrest between
two groups. (GV = 21.78%, M-I = 10.67%, P < 0.05)
4.2. Ultrastructure of M-II Oocyte
In the control M-II oocytes, the zona pellucida (ZP) consisted of closely packed electron dense fibrilar material.
The perivitelline space (PVS) was uniform and scarce debris was usually found in it. The oolemma was continuous, and provided with numerous long-thin microvilli
were uniformly distributed on the oolemma, except in
the zone of polar body extrusion where microvilli were
absent. Another feature was the presence of round cortical granules with an electron dense appearance that was
located just beneath the oolemma (Figures 1 A and D).
The majority of the oocyte organelles consisted of aggregates of smooth endoplasmic reticulum (SER) surrounded by spherical or elliptical shaped mitochondria
(M-SER aggregates). Small and large, spherical M-SER
aggregates were found randomly distributed in the ooplasm (Figure 2 A). Small mitochondria-vesicle complexes (M-VC) were also observed (Figure 2 D). The arc-like
or transverse cristae of mitochondria were irregularly
placed on the periphery and parallel to the outer mitochondrial membrane. These cristae contained a moderIran Red Crescent Med J. 2013;15(12):e7379
Shahedi A. et al.
ately electron dense matrix (Figures 2 A and D).
Figure 1. General Fine Structure and Organelle Microtopography are shown by Transmission Electron Microscopy.
In control (A, D), GV (B, E) & MI (C, F) stage oocyte after IVM. Microvilli are numerous and long on the oolemma of oocytes. A rim of electron-dense cortical
granules (arrows) is seen just beneath the oolemma of oocytes. ZP = zona pellucida; mv = microvilli; CG = cortical granules; O = oocyte; PVS = perivitelline
space; PB = polar body
Figure 2. Control Oocyte (A, D), GV (B, E) & MI (C, F) Stage Oocyte after IVM.
Mitochondria are generally rounded and provided with few peripheral or transverse cristae. Dumbbell shaped, possibly dividing mitochondria can be
occasionally find in the ooplasm (C). Migrating cortical granule is seen in C (arrow). Voluminous aggregates between mitochondria and elements of
smooth endoplasmic reticulum are seen (A-C). Note the presence of complexes between mitochondria and vesicles of smooth endoplasmic reticulum in
D - F (arrows). Migrating cortical granules are seen in E (arrow heads). SER = smooth endoplasmic reticulum; M = mitochondria
4.3. Ultrastructure of GV and MI Oocytes after IVM
The ultra-structural characteristics of IVM oocytes had
some similarity to that of the control M-II oocytes. HowIran Red Crescent Med J. 2013;15(12):e7379
ever, the most notable feature of the matured oocytes after IVM was the presence of numerous large M-VC within
their cytoplasm as compared to in-vivo matured oocytes
(Figures 2 E and F). In general, the population of M-VC was
3
Shahedi A. et al.
more distributed in GV when compared with MI oocytes
(Figure 2 E). Small and large, spherical M-SER aggregates
were found randomly distributed in the ooplasm (Figure
2 B and C). Other organelles, such as mitochondria, cortical granules, microvilli, PVS and ZP were similar between
groups (Figures 1 E and F, 2B and C).
5. Discussion
In ovarian stimulation cycles, maturation of 85-90% oocytes may be triggered by the administration of hCG 36
h before oocyte retrieval. In conventional IVF cycles, the
remaining immature oocytes at the time of collection
are not suitable for ICSI. These immature oocytes, upon
patient’s agreement, are generally destined to research
(5). Why these oocytes fail to complete nuclear maturation remains speculative. It has been proposed that the
follicles may be resistant or less responsive to hormonal
stimulation (13) or they simply may be at an earlier developmental phase (14). In this study, we demonstrated
that rate of maturation of GV oocytes was lower than M-I
oocytes (61.38% vs. 73.78%). This is, probably due to the differences at the oocyte developmental stage at the time
of oocyte collection. Previous studies indicated the IVM
rate as low as 37% for GV oocytes collected in stimulated
cycles (5, 14). These results are not comparable with our
findings (61.38%), which is likely due to the differences at
duration of maturation and composition of IVM medium. Previous studies, investigating the clinical outcome
of immature oocytes retrieved from stimulated ICSI cycles, have shown that early matured oocytes yield significantly higher fertilization and developmental potential
than their later matured counterparts (15). Tan et al. (4)
reported the maturation rate of 70.8% for human M-I oocytes collected from stimulated cycles, which is similar to
our findings (1). Despite the fact that implantation rate
per transferred embryo using in-vitro matured oocytes
doesn’t yet reach the outcomes of conventional IVF (16),
this technique is already offered to patients at high risk of
developing hyper-stimulation syndrome or for fertility
preservation. In vivo and within primary oocytes, cellular
components of endoplasmic reticulum (ER), mitochondria and Golgi complexes are seen around the nucleus
(17). Once, meiosis starts, organelles distribute throughout the cell. As reported, no ultra-structural differences
could be observed between spontaneously ovulated and
ovulation-induced oocytes (18). In this study, numerous
large M-VC observed in the IVM oocytes that may be sign
of prolonged stay in vitro (in vitro aging) and could be
potential determinants of a reduced oocyte competence
for fertilization. M-SER aggregates are currently considered as a precursor of mitochondria-vesicle complexes
(M-VC) and may play a role in the production of materials useful at fertilization and / or in rapid neoformation
of membranes during early embryogenesis (19). M-SER
aggregates may also regulate local concentration of free
calcium and ATP production, thus acting on different cel4
lular activities including mediation of calcium–dependent signal transduction pathways at fertilization (20).
The ultra-structural alterations in the arrangement of
M-SER aggregates could be, among other factors, potential determinants of a reduced oocyte competence for
fertilization, possibly due to disturbances in calcium homeostasis. Variations in shape and size of M-SER, without
ultra-structural changes in both SER tubules and mitochondria, could be related to different morphodynamic
stages of these elements (12).
In our study, cortical granules were normal and no
ultra-structural changes were demonstrated between
control and IVM groups. Wessel et al. (21) reported that
cortical granules are Golgi-derived; membrane bound
spherical or slightly ellipsoid organelles formed during
the early stages of oocyte and at maturation (21). Exocytosis of the cortical granule content into PVS immediately
after oocyte fertilization lead to changing of the ZP glycoproteins, establishing the block to polyspermy. This
study showed that in all groups, oocytes were surrounded by an intact, continuous oolemma, provided with
long microvilli projecting into a PVS with regular shape
and width. It has been reported that proper distribution
of microvilli on the oocyte surface is determinant for the
spermatozoon-oocyte fusion (22).
In conclusions, immature human oocytes at different
stages of development subjected to the IVM showed minimum of alteration in cytoarchitecture. However, ultra
structural changes of M-VC following IVM emphasize the
need for further research in order to refine culture conditions and improve the implantation rate of in vitro matured oocytes.
Acknowledgements
The authors would like to thank the colleagues who
helped with experiments and the data presented in this
article. The present study was supported by grants from
the research & clinical center for infertility.
Authors’ Contribution
All authors have participated in the study.
Funding Support
The study is self-funded.
Financial Disclosure
There is no conflict of interest.
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